Efficient In Vitro and In Vivo Activity of Glyco

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RESEARCH ARTICLE

Efficient In Vitro and In Vivo Activity of Glyco-Engineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3 Tsepo Lebiletsa Tsekoa1, Therese Lotter-Stark1, Sindisiwe Buthelezi1, Ereck Chakauya1, Stoyan H. Stoychev1, Claude Sabeta2, Wonderful Shumba2, Baby Phahladira2, Steve Hume4, Josh Morton4, Charles E. Rupprecht5, Herta Steinkellner6, Michael Pauly3, Larry Zeitlin3, Kevin Whaley3, Rachel Chikwamba1*

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1 Biosciences Unit, Council for Scientific and Industrial Research, Pretoria, South Africa, 2 ARCOnderstepoort Veterinary Institute, Onderstepoort, South Africa, 3 Mapp Biopharmaceutical, San Diego, California, United States, 4 Kentucky Bioprocessing, Owensboro, Kentucky, United States, 5 The Wistar Institute, Philadelphia, Pennsylvania, United States, 6 Department of Applied Genetics and Cell Biology, University of Natural Resources and Life Sciences, Vienna, Austria * [email protected]

OPEN ACCESS Citation: Tsekoa TL, Lotter-Stark T, Buthelezi S, Chakauya E, Stoychev SH, Sabeta C, et al. (2016) Efficient In Vitro and In Vivo Activity of GlycoEngineered Plant-Produced Rabies Monoclonal Antibodies E559 and 62-71-3. PLoS ONE 11(7): e0159313. doi:10.1371/journal.pone.0159313 Editor: Gunnar F Kaufmann, The Scripps Research Institute and Sorrento Therapeutics, Inc., UNITED STATES Received: January 16, 2016 Accepted: June 30, 2016 Published: July 18, 2016 Copyright: © 2016 Tsekoa et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: LZ and KW are owners of Mapp Biopharmaceutical, a commercial company. MP is employed by Mapp Biopharmaceutical, while JM and SH are employed by Kentucky Bioprocessing, a commercial company. The authors thank the National Research Foundation (SB) (www.nrf.ac.za), Technology Innovation Agency (RC) (www.tia.org.za), the Agricultural Research Council Rabies Diagnostic Cost Centre (15/04/P001) (CS) and Council for

Abstract Rabies is a neglected zoonotic disease that has no effective treatment after onset of illness. However the disease can be prevented effectively by prompt administration of post exposure prophylaxis which includes administration of passive immunizing antibodies (Rabies Immune Globulin, RIG). Currently, human RIG suffers from many restrictions including limited availability, batch-to batch inconsistencies and potential for contamination with bloodborne pathogens. Anti-rabies monoclonal antibodies (mAbs) have been identified as a promising alternative to RIG. Here, we applied a plant-based transient expression system to achieve rapid, high level production and efficacy of the two highly potent anti-rabies mAbs E559 and 62-71-3. Expression levels of up to 490 mg/kg of recombinant mAbs were obtained in Nicotiana benthamiana glycosylation mutants by using a viral based transient expression system. The plant-made E559 and 62-71-3, carrying human-type fucose-free Nglycans, assembled properly and were structurally sound as determined by mass spectrometry and calorimetric density measurements. Both mAbs efficiently neutralised diverse rabies virus variants in vitro. Importantly, E559 and 62-71-3 exhibited enhanced protection against rabies virus compared to human RIG in a hamster model post-exposure challenge trial. Collectively, our results provide the basis for the development of a multi-mAb based alternative to RIG.

Introduction Rabies is a zoonotic viral disease that continues to have no effective treatment after onset of symptoms. Typically, infection occurs after a bite from an infected animal, principally the domestic dog. Other animal species, notably wild carnivores and bats, serve as reservoirs of the

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Scientific and Industrial Research (SA) (TT, RC) (www.csir.co.za) for funding support. The funders provided support in the form of salaries for authors SB, TT and RC, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section. Competing Interests: LZ and KW are owners of Mapp Biopharmaceutical, a commercial company. MP is employed by Mapp Biopharmaceutical, while JM and SH are employed by Kentucky Bioprocessing, a commercial company. This does not alter the authors’ adherence to PLOS ONE policies on sharing data and materials.

rabies virus. Post exposure prophylaxis (PEP) is highly effective when administered promptly. The recommended PEP regimen includes immediate administration of Rabies Immune Globulin (or RIG) from pooled sera taken from hyper-immunized horses (ERIG) or humans (HRIG), as well as vaccination with inactivated Rabies Vaccine and thorough wound cleansing [1, 2]. The majority of the approximately 55,000–70,000 annual human rabies fatalities occur in the developing world, yet access to RIG for adequate PEP is still poor in those countries due to affordability and availability [3, 4]. Both HRIG and ERIG are often in short supply due to the exponential increase in demand for PEP in recent years. In addition, RIG suffers shortcomings such as inconsistency between batches, potential for contamination with blood-borne diseases and, in particular for ERIG, occasional adverse allergic reactions such as serum sickness or anaphylactic shock are observed [5]. For these reasons, an international drive to develop alternative PEP biologics, led by the World Health Organisation (WHO), is underway and replacement of RIG with a safer, efficacious and potentially more economical alternative biologic remains a priority. With the involvement of the WHO Collaborating Centres for Rabies Surveillance and Research, several mouse-derived monoclonal antibodies (mAbs) with rabies virus neutralizing activity have been identified [6]. These mAbs have been targeted for future development to replace RIG as components of a safer new generation for PEP that is affordable for cost-sensitive markets. Clearly, mAbs have several advantages over RIG including better consistency, improved safety, and with humanization, improved tolerance in patients [7, 8]. Alternatively, due to the specificity of individual neutralizing mAbs for different epitopes on the rabies virus glycoprotein, mAb-based products may have limited potential unless they are formulated as a cocktail to avoid virus escape, improve potency and to broaden their viral neutralization breadth, because there is no single pan-reactive mAb against such diverse lyssaviruses documented to date [9]. Given the specificity of mAb 62-71-3 for antigenic site I, all proposed cocktails from the WHO program so far are envisaged to include 62-71-3, and one of mAbs E559.9.14, M7275-1, M777-16-3 or 1112–1. Among these, mAb E559 has a broad rabies virus isolate breadth of specificity and potency [6, 9]. The current study describes the plant-based recombinant expression, purification, structural and functional characterisation (in vitro and in vivo) of humanized anti rabies mAbs E559 and 62-71-3. mAbs were expressed in ΔXT/FT, a Nicotiana benthamiana mutant supporting the synthesis of glycan-optimized fucose-free mAbs [10]. Transient expression in plants using virus based vectors was selected as a highly scalable, rapid production alternative to mammalian cell (e.g., CHO cell) culture-based production. Plant expressed mAbs efficiently neutralized a set of virus strains in a cell based Rapid Fluorescent Focus Inhibition test (RFFIT) assay. Moreover, mAbs exhibited enhanced in vivo potency compared to HRIG as determined by a hamster viral challenge model.

Results/Discussion Recombinant Expression of full length chimeric IgG mAb E559 and 6271-3 in Nicotiana benthamiana Variable domains from light and heavy chain (VH and VL) from murine mAbs E559 and 6271-3 were fused to the constant domain (CH and CL) from human IgG1. These chimeric constructs were plant codon-optimised light chain (LC) and heavy chain (HC) expression constructs with two different signal peptides. These chimeric Ab genes were cloned into two noncompeting plant viral vectors, tobacco mosaic virus (TMV) and potato virus X (PVX) backbones [11]. The LC and HC vectors were combined and infiltrated into glycoengineered

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Table 1. MAb E559 and 62-71-3 expression levels obtained using various combinations of either PVX or TMV-based expression vectors with either the murine (m) or rice alpha amylase (r) signal peptide. Molecule

Vector combinations

Green tissue (g)

Expression (μg/g)

E559

TMV-rHC +PVX-rLC

10

17

E559

TMV-mHC + PVX-mLC

10

456

E559

PVX-rHC + TMV-rLC

10

67

E559

PVX-mHC + TMV-mLC

10

87

62-71-3

TMV-rHC + PVX-rLC

10

339

62-71-3

TMV-mHC + PVX-mLC

10

455

62-71-3

PVX-rHC + TMV-rLC

10

106

62-71-3

TMV-mHC + PVX-mLC

10

278

E559

TMV-mHC + PVX-mLC

1000

349

62-71-3

TMV-mHC + PVX-mLC

1000

493

doi:10.1371/journal.pone.0159313.t001

ΔXTFT Nicotiana benthamiana plants [10] that were monitored for expression of assembled IgG. For both mAbs E559 and 62-71-3 the highest expression levels (456 mg/kg and 455 mg/kg respectively) determined by ELISA were attained during initial expression evaluation using the murine signal peptide in the TMV-HC and the PVX-LC combination (Table 1). The expression levels remained constant upon upscaling the procedure in a highly regulated contract manufacturer environment. These expression levels are higher than that observed when antibodies were expressed using transgenic approaches [12, 13]. These data provide a suitable basis for modelling a scaled-up, economically viable manufacturing process.

Analytical characterisation of plant-produced chimeric mAbs E559 and 62-71-3 Most mAbs can be subject to many potential modifications, including proteolytic clipping, glycosylation, deamidation and oxidation, all of which can affect their efficacy and formulation stability [14]. Therefore, it is important to comprehensively characterise biochemical properties and molecular structures. Protein A-purified mAbs were characterized by SDS-PAGE under reduced conditions (Fig 1). Both E559 and 62-71-3 heavy chain (HC) bands migrated to their predicted MWs (50 and 25 kDa). Using online LC-ESI-TOF MS was established to elucidate the identity of the mAbs. The deconvoluted multiply charged spectrum of reduced E559 LC is shown in Fig 2(A). The major peak in the spectrum matched the theoretical LC MW of 23,505.87Da. The peaks at 24,398Da, 24,601Da and 24,805Da matched the water-eliminated complex glycans GlcNac2Man3, GlcNac2Man3GlcNac1 and GlcNac2Man3GlcNac2, respectively. In the HC region of E559 only the glycosylated species were observed whilst the native peak at 49,280.18Da was below the limit of detection (Fig 2B). The deconvoluted multiply charged mass spectrum of reduced 62-71-3 indicated one major peak in the region of the light chain (LC) at 23,670 Da (Fig 3, inset) and no peaks indicative of glycosylation. A putative glycosylation peak was detected at 50,333 Da, 1,298 Da from the 6271-3 HC of 49,035Da (Fig 3). The mass difference of 1,298Da was indicative of the complex sugar GlcNac2Man3GlcNac2, with a single water molecule eliminated. Several mAbs, whose N-glycans lack fucose, have been demonstrated to enhance in vivo efficacy in different models of viral infection [15–17] due to increased ADCC activity. In addition, afucosylated therapeutic anti-cancer antibodies can exhibit superior in vitro and in vivo efficacy [18, 19]. For these reasons, the anti-rabies mAbs were expressed in the ΔXTFT Nicotiana

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Fig 1. SDS-PAGE analysis of purified E559 and 62-71-3 mAbs. The middle lane was a PageRuler Prestained Protein Ladder, with the sizes indicated in kDa. The numbered E559 and 62-71-3 bands (1–5) were used for further analysis. doi:10.1371/journal.pone.0159313.g001

benthamiana host, which is a glycosylation mutant synthesizing predominantly fucose free GnGn glycan structures [10]. To elucidate site-specific glycosylation of the antibodies, respective glycopeptides of HC and LC were analysed by LC-ESI-MS after protein tryptic digest [20]. The glycopeptide profiles of 62-71-3 and E559 HCs were identical and exhibited a single dominant N-glycan species i.e. GlcNac2Man3GlcNac2 (referred to as GnGn) (Fig 4). The major glycan species on the LC of E559 refers to GlcNac2Man3GlcNac1 (GnM). As expected, the LC of 62-71-3 yielded no glycopeptides (data not shown) corroborating findings from the intact LCMS analyses (Fig 3). Our data show that the molecular weight of each mAb precisely matched the mass expected from the deduced amino acid sequence and had the expected glycan profile.

Secondary and Tertiary structural characterization To determine structural integrity of plant produced E559 and 62-71-3 secondary and tertiary structure of E559 62-71-3 were determined using Circular Dichroism Spectroscopy (CD: secondary structure probe) and Fluorescence Spectroscopy (FS: tertiary structure probe). The spectra of native mAb molecules were expected to be dominated by β-sheets with few α-helix conformations found in typical antibodies [21]. The Far-UV CD spectra of the two mAbs were closely related with both mAbs exhibiting minima in the 217 nM region indicating that the secondary structural content is indeed dominated by β-sheets (Fig 5). The tertiary and quaternary structures of E559 and 62-71-3 were compared using FS. Both excitation at 280 nm (combined excitation of Trp and Tyr residues) and 295 nm (selective excitation of Trp

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Fig 2. Deconvoluted spectrum of intact, reduced E559 LC (A) and intact, reduced E559 HC (B).Theoretical molecular weights for LC and HC indicated. Detected N-linked glycoforms are shown. The N-glycan nomenclature used was from www.proglycan.com. doi:10.1371/journal.pone.0159313.g002

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Fig 3. Deconvoluted spectrum of intact, reduced 62-71-3 HC. The inset shows the zoomed-in LC region with theoretical molecular weights for LC and HC indicated. Detected N-linked glycoforms are shown. The N-glycan nomenclature used was from www.proglycan.com. doi:10.1371/journal.pone.0159313.g003

residues) were used to monitor global differences between the two mAbs. The E559 has 19 Tyr and 10 Trp residues in the HC and 10 Trp and 2 Tyr in the LC. The 62-71-3 mAb has 18 Tyr and 9 Trp residues in the HC and in the LC it has 10 Trp and 2 Tyr, with residues distributed in a similar manner. At both excitation wavelengths, the λemm max of E559 was shifted to longer wavelengths. This observation indicated a more exposed environment of Trp and Tyr residues

Fig 4. N-linked glycans on the anti-rabies mAbs. N-glycosylation profile from E559 HC and LC and from 62-71-3 HC as determined by LC-ESI-MS of glycopeptides obtained upon trypsin digestion. Numbers represent the presence of the different glyco-species in percent of total glycan. The N-glycan nomenclature used was from www.proglycan.com. doi:10.1371/journal.pone.0159313.g004

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Fig 5. Far-UV CD spectra of humanised 62-71-3 (black) and E559 (blue) mAbs. doi:10.1371/journal.pone.0159313.g005

for E559 suggesting a more loosely packed quaternary conformation as compared to 62-71-3 (Fig 6).

Thermal stability To determine which mAb is more vulnerable to heat induced degradation, the thermal stability was measured by monitoring changes in secondary structural content [22]. The samples were heated continuously at 5°C per minute from 25 oC to 90 oC and far-UV spectra were measured in the region 180–260 nm. Since both mAb structures are dominated by β-sheets, changes at the 217 nm minima, indicative of β-sheet content, was monitored [21]. Differences were observed from 50–55 oC indicating possible rearrangement in secondary structural content in the case of E559. On the other hand, changes in β-sheet content, for 62-71-3, were only observed above 65 oC (Fig 7). Treatment with antibody combinations can be challenging

Fig 6. Fluorescence emission spectra of 62-71-3 (black) and E559 (blue). The mAbs were excited at 280 nm (A) and 295 nm (B). λemm max for each mAb is marked with a dotted line. doi:10.1371/journal.pone.0159313.g006

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Fig 7. Changes in the β-sheets content of 62-71-3 (black) and E559 (blue) mAbs with increasing temperature. doi:10.1371/journal.pone.0159313.g007

because in spite of their common structure, individual mAbs often have unique and unpredictable responses to their environment related to their stability [23]. This will also be the case with the envisaged E559 / 62-71-3 cocktail where the data suggest E559 is less thermostable than 6271-3.

Neutralisation potency of plant-made E559 and 62-71-3 The two murine mAbs E559 and 62-71-3 were reported to recognise two complementary sites on the rabies virus glycoprotein [6, 9]. In the current study, it was important to confirm that the chimeric, plant-made versions were effective and secondly, to determine their complimentary efficacy or neutralisation pattern against rabies virus isolates in the context of their potential future application as a cocktail rabies PEP biopharmaceutical. To test the breadth and coverage of the plant-made E559 and 62-71-3 on 31 laboratory and field isolates of rabies virus, a modified Rapid Fluorescent Focus Inhibition test (RFFIT) was conducted and the neutralisation activity was determined as a 50% end point neutralisation (reciprocal titre). Table 2 and Fig 8 show the neutralisation activities. As expected the mAbs neutralised the laboratory strain CVS-11 but had varied neutralisation activity levels on field isolates of rabies virus from different parts of the world. MAb E559 neutralised all the isolates except Bat 3860, Fox (TX), Dog (Philipine and Argentine), RVHN and Mongoose (South Africa), while both mAbs were not active against Skunk (CA) and Bat Lasiurus cinereus (NY). Because of the difficulty of transferring isolates across national borders, the sample viruses had only two isolates from African countries. The Dog (Tunisia) isolate was neutralised by both mAbs, while mAb 62– 713 neutralised the Mongoose (South Africa) isolate while mAb E559 could not. Antibody E559 binds to the discontinuous antigenic sites II while 62-71-3 binds to antigenic site I of the Rabies virus glycoprotein (RVG). As these antibodies have different binding sites, they provide the capacity to simultaneously bind RVG. Use of these two Abs in a cocktail will enhance the neutralization of wider spectra of rabies viruses and reduce the chances of virus escape [9].

In vivo efficacy The in vivo potency of the plant-produced mAbs was tested in a challenge experiment with female Syrian hamsters infected with CVS-11 (Fig 9). The infected control group did not survive beyond 14 dpi confirming the lethality of the viral inoculum. Animals receiving treatment were administered 2 International Units (IU) of mAb or HRIG. Animals treated with the plant-made mAb E559 (Group 2) showed 100% protection at 14 dpi, slightly higher than that

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Table 2. Fifty percent end-point neutralisation activity (reciprocal titre) of E559 and 62-71-3 in a modified Rapid Fluorescent Focus Inhibition Test (RFFIT). Rabies Virus

Rabies Virus

VNA titres

VNA titres

62-71-3

E559

CVS-11

280

54

Raccoon

62-71-3 95

E559 8

Dog, Argentina

625